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Author: Christos G. Tsokos
Publisher:
ISBN:
Category :
Languages : en
Pages : 127
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Book Description
Cellular asymmetry is critical to the generation of complexity in both metazoans and many microbes. However, several molecular mechanisms responsible for translating asymmetry into differential cell fates remain unknown. Caulobacter crescentus provides an excellent model to study this process because every division is asymmetric. One daughter cell, the stalked cell, is sessile and commits immediately to S phase. The other daughter, the swarmer cell, is motile and locked in G1. Cellular differentiation requires asymmetric distribution or activation of regulatory factors. In Caulobacter, the master cell cycle regulator CtrA is selectively activated in swarmer cells, deactivated in stalked cells, and reactivated in predivisional cells. CtrA controls DNA replication, polar morphogenesis and cell division, and its cell-type and cell cycle-specific regulation is essential to the life cycle of Caulobacter. In swarmer cells, activated CtrA binds to the origin of replication and holds cells in G1. In stalked cells, CtrA deactivation allows for the initiation of DNA replication. Finally, in predivisional cells, CtrA is reactivated and acts as a transcription factor for>100 genes including those involved in polar morphogenesis and cell division. CtrA regulation is determined by the polarly localized histidine kinase CckA, but how CckA is differentially regulated in each cell type and why activity depends on localization are unknown. This thesis demonstrates that the unorthodox kinase DivL promotes CckA activity and that the phosphorylated regulator DivK inhibits CckA by binding to DivL. Differential cellular fates are achieved by regulating the phosphorylation state of DivK. In swarmer cells, DivK is dephosphorylated, thereby activating CckA and arresting the cells in G1. In stalked cells, phosphorylated DivK inactivates CckA, thus allowing for DNA replication initiation. Paradoxically, in predivisional cells, while phosphorylated DivK levels remain high, CckA is reactivated to initiate cellular division and morphogenesis. CckA activation in this cell type relies on polar localization with a DivK phosphatase. Localization thus creates a protected zone for CckA within the cell, without the use of membrane-enclosed compartments. These results reveal the mechanisms by which CckA is regulated in a cell-type-dependent manner. More generally, these findings reveal how cells exploit subcellular localization to orchestrate sophisticated regulation.
Author: Christos G. Tsokos
Publisher:
ISBN:
Category :
Languages : en
Pages : 127
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Book Description
Cellular asymmetry is critical to the generation of complexity in both metazoans and many microbes. However, several molecular mechanisms responsible for translating asymmetry into differential cell fates remain unknown. Caulobacter crescentus provides an excellent model to study this process because every division is asymmetric. One daughter cell, the stalked cell, is sessile and commits immediately to S phase. The other daughter, the swarmer cell, is motile and locked in G1. Cellular differentiation requires asymmetric distribution or activation of regulatory factors. In Caulobacter, the master cell cycle regulator CtrA is selectively activated in swarmer cells, deactivated in stalked cells, and reactivated in predivisional cells. CtrA controls DNA replication, polar morphogenesis and cell division, and its cell-type and cell cycle-specific regulation is essential to the life cycle of Caulobacter. In swarmer cells, activated CtrA binds to the origin of replication and holds cells in G1. In stalked cells, CtrA deactivation allows for the initiation of DNA replication. Finally, in predivisional cells, CtrA is reactivated and acts as a transcription factor for>100 genes including those involved in polar morphogenesis and cell division. CtrA regulation is determined by the polarly localized histidine kinase CckA, but how CckA is differentially regulated in each cell type and why activity depends on localization are unknown. This thesis demonstrates that the unorthodox kinase DivL promotes CckA activity and that the phosphorylated regulator DivK inhibits CckA by binding to DivL. Differential cellular fates are achieved by regulating the phosphorylation state of DivK. In swarmer cells, DivK is dephosphorylated, thereby activating CckA and arresting the cells in G1. In stalked cells, phosphorylated DivK inactivates CckA, thus allowing for DNA replication initiation. Paradoxically, in predivisional cells, while phosphorylated DivK levels remain high, CckA is reactivated to initiate cellular division and morphogenesis. CckA activation in this cell type relies on polar localization with a DivK phosphatase. Localization thus creates a protected zone for CckA within the cell, without the use of membrane-enclosed compartments. These results reveal the mechanisms by which CckA is regulated in a cell-type-dependent manner. More generally, these findings reveal how cells exploit subcellular localization to orchestrate sophisticated regulation.
Author: Adam Michael Perez
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
The subcellular localization of different cell fate determinants at the cell poles serves to enable asymmetric cell division in many diverse cell types. The bacterium Caulobacter crescentus carries out an asymmetric cell division in each cell cycle. Key to the generation and maintenance of asymmetry in Caulobacter is the localization of two distinct sets of two-component signaling proteins to opposing cell poles. The asymmetric localization of these proteins ensures differential transcriptional readouts of the genome to produce two daughter cells with different cell fates upon the completion of cell division. The molecular mechanisms that are utilized to recruit these proteins to the pole remain ill defined. In this thesis work, I present evidence detailing the assembly of a polar protein complex that drives the asymmetric cell cycle of Caulobacter. Key to the recruitment of multiple polar proteins is the PopZ protein that forms a polymeric matrix at the cell pole. As it is one of the first proteins known to localize to the Caulobacter cell pole and is required for localization of at least 10 known polar proteins, it is critical to know how PopZ functions as a polar organizer. To understand how polar organizing centers are established by PopZ, I generated a set of mutated PopZ proteins and investigated the mutant strains for defects in sub-cellular localization and recruitment activity. I identified a domain within the C-terminal 76 amino acids of PopZ that is necessary and sufficient for accumulation as a single subcellular focus, and a 23 amino acid domain at the N-terminus that is necessary for bipolar targeting. Mutations in either domain caused defects in the recruitment of other factors to the cell poles, indicating a role for dynamic PopZ localization in polar organization. Mutations in the C-terminal domain also blocked discrete steps in the PopZ matrix assembly pathway. Biophysical analysis of purified wildtype and assembly-defective mutant proteins revealed that the PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into long filaments, which are important for subcellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter. Downstream of PopZ, localization of the histidine kinase DivJ specifically to the stalked cell pole is critical for defining the stalked cell fate. As the Caulobacter cell cycle progresses, both the flagellum-bearing pole and the new stalked pole have a PopZ polymeric matrix, but the new stalked pole acquires the SpmX protein and the DivJ histidine kinase. Using both a heterologous in vivo expression system and binding assays with purified proteins, I determined the pathway of stalked pole complex formation. I demonstrate that SpmX, which is synthesized only at the swarmer to stalked cell transition, binds directly to PopZ via its lysozyme-like domain. Subsequently, newly synthesized DivJ binds directly to SpmX. The positioning of DivJ at one cell pole spatially restricts this histidine kinase so that upon division it is sequestered to the progeny stalked cell where it mediates stalked cell fate determination. Analysis of SpmX truncations and amino acid substitutions revealed that the additional regions of the SpmX protein, including a proline rich domain and the transmembrane domains, contribute to the asymmetric localization of SpmX, and consequently DivJ. Mistimed overproduction of SpmX resulted in the initiation of lateral growth zones providing insight into the function of the SpmX protein as a mediator of the three dimensional organization of the cell and, via DivJ, the maintenance of asymmetry. This thesis work utilizes a combination of biochemistry, cell biology, and molecular biology to detail the assembly of a protein complex that serves to maintain and generate cellular asymmetry in Caulobacter. The insights gained into the mechanisms that drive formation of this complex have broad implications in the understanding of subcellular protein localization in other bacteria, as well as in the understanding of cellular asymmetry in all kingdoms of life.
Author: Elaine Benner Shapland
Publisher:
ISBN:
Category :
Languages : en
Pages : 146
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Book Description
How bacteria control their shape and division was one of the first topics investigated with molecular biology, and many unanswered questions remain today. This dissertation research used the model organism Cualobacter crescentus to investigate how phospho-signaling controls asymmetric cell division, and how those signals are initiated and regulated. Most signaling in bacteria is achieved through two component systems (TCS), which are comprised of a histidine kinase and a response regulator. The downstream effects of response regulator activation have been well documented and can affect gene transcription, protein interactions or enzyme activity. However, very little is known about how histidine kinases are activated. Caulobacter uses TCS to control its asymmetric cell division and differentiation, but the events that initiate the cell cycle and the ability of an outside signal to impinge upon cell cycle progression remain unknown. Using three different methods, I have been able to shed light on signaling and cell cycle progression in Caulobacter crescentus. I have developed a tool to determine which proteins and conditions activate histidine kinases. I have shown that an outside environmental signal can feed into the TCS controlling cell cycle progression. I have also shown that a protein similar to a eukaryotic tyrosine phosphatase controls membrane integrity and morphology and is essential for viability in Caulobacter.
Author: Stephen Carl Smith
Publisher:
ISBN:
Category :
Languages : en
Pages : 102
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Book Description
Caulobacter crescentus is a powerful model organism for understanding cellular differentiation, cell polarity and cell cycle regulation in bacteria. An elaborate network of two-component signaling proteins works to orchestrate the developmental program that characterizes the Caulobacter cell cycle. The essential DNA-binding response regulator CtrA is at the center of this regulatory scheme and acts to control the transcription of>100 genes that are required for cell cycle progression, motility, DNA methylation, morphology and other processes. Because CtrA also inhibits chromosome replication at specific stages of the Caulobacter cell cycle, its activity must be temporarily eliminated in order for DNA replication to occur. Inactivation of CtrA is achieved though dephosphorylation and regulated degradation by the broadly conserved energy-dependent protease ClpXP. In this dissertation, I analyze the roles of three proteins that are required for CtrA degradation in living cells. These are a single domain response regulator CpdR, a protein with no predicted function, RcdA, and a cyclic diguanylate (cdG)-binding protein, PopA. Structure-directed mutagenesis of RcdA was used to probe RcdA function. Results from these studies undermine the prevailing model for RcdA function, which suggest that RcdA does not participate directly in delivering CtrA to ClpXP, but instead acts simply as a localization factor increasing the concentration of CtrA at the cell pole where the protease is located. Additionally, I reconstituted the regulated proteolytic reaction in vitro and probed the role of all three accessory proteins and the small molecule cdG in promoting CtrA degradation. Although ClpXP alone is known to degrade CtrA in vitro, I observed a dramatic acceleration of proteolysis in the presence of the accessory proteins and cdG. This accelerated proteolysis was characterized by a nearly 10-fold reduction in the KM of the reaction, which is consistent with predictions for an adaptor mediated mechanism. I began to characterize protein-protein interactions within the proteolytic complex using in vivo and in vitro techniques. These experiments demonstrate that CtrA interacts directly with PopA in a cdG-dependent fashion. CtrA also interacts directly with RcdA and with ClpX. The CtrA-PopA(cdG) and CtrA-RcdA interactions are weakened or abolished by mutations in the receiver domain of CtrA that slow its proteolysis in vivo. We propose a mechanism in which CtrA forms a ternary complex with PopA and RcdA in response to rising cdG concentrations in the cell. In this complex, PopA and RcdA act as a multi-protein adaptor complex to enhance the delivery of CtrA to the catalytic pore of ClpX. CpdR is required for accelerated CtrA proteolysis, but its precise role is still unknown. The accessory proteins were able to stimulate CtrA degradation even in the presence of a DNA fragment containing a CtrA binding site, which is known to inhibit CtrA proteolysis. Future work will determine if the accessory factors prevent the formation of inhibitory CtrA-DNA complexes or actively disassemble them. This dissertation alters the concept of proteolytic adaptors to include multi-protein complexes and expands the range of mechanisms by which proteolytic adaptors are controlled to include direct regulation by the small molecule cdG.
Author: Haibi Wang
Publisher:
ISBN: 9781658425261
Category : Cell cycle
Languages : en
Pages : 149
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Book Description
Cell division and differentiation are complex biological phenomena that occur in all kingdoms of life. Understanding the molecular mechanisms that underlie these complex processes often requires the study of experimentally tractable model organisms. As a Gram-negative bacteria with less than four thousand genes, Caulobacter crescentus exhibits cell differentiation, highly regulated chromosome replication and segregation, and asymmetric cell division with every turn of the cell cycle. To achieve these behaviors, Caulobacter utilizes spatial control mechanisms such as sub-cellular compartmentalization and protein localization. The cell poles are particularly enriched for cell cycle regulatory proteins. Many of these proteins are localized by the hub protein PopZ, which forms a three-dimensional scaffold that also aids in chromosome segregation. The PopZ scaffold also includes proteolysis activity, which regulates cell cycle progression in a manner that is analogous to well-known eukaryotic systems. In this dissertation, I characterized an evolutionarily conserved protein of unknown function, which is now named SpbR (Swarmer pole blocking factoR). SpbR is a pole-localized protein that has co-evolved with PopZ and other polar proteins. Strikingly, SpbR over-production exhibited a severe chromosome segregation phenotype, in which the newly replicated centromere failed to travel across the cell to its normal destination at the opposite pole. SpbR overproduction results in its accumulation at the old pole, where it physically interacts with PopZ. This prevents the relocation of PopZ to the new pole, thereby eliminating a positional cue for centromere translocation. Consistent with this, the centromere translocation phenotype of SpbR overproducing cells is further enhanced in genetic backgrounds that accumulate higher SpbR or reduce chromosome segregation activity. We find that pole-localized SpbR is normally cleared by proteolysis before the time of chromosome segregation, indicating that SpbR turnover is part of the cell cycle-dependent program of polar development.
Author:
Publisher: Academic Press
ISBN: 0323915426
Category : Science
Languages : en
Pages : 838
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Book Description
Pseudokinases, Volume 667, the latest release in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting interesting chapters, including the Production and Purification of the PEAK pseudokinases for structural and functional studies, Structural biology and biophysical characterization of Tribbles pseudokinases, Detecting endogenous TRIB protein expression and its downstream signaling, Analysis of human Tribbles 2 pseudokinase, Expression, purification and examination of ligand-binding to IRAK pseudokinases, Characterization of pseudokinase ILK-mediated actin assembly, Biochemical examination of Titin pseudokinase, Approaches to study pseudokinase conformations, CRISPR editing cell lines for reconstitution studies of pseudokinase function, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in Methods in Enzymology serials Includes the latest information on Pseudokinases
Author: Emanuele Biondi
Publisher: Springer Nature
ISBN: 3030906213
Category : Science
Languages : en
Pages : 305
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Book Description
This book provides a comprehensive overview of the cell cycle regulation and development in Alphaproteobacteria. Cell cycle and cellular differentiation are fascinating biological phenomena that are highly regulated in all organisms. In the last decades, many laboratories around the world have been investigating these processes in Alphaproteobacteria. This bacterial class comprises important bacterial species, studied by fundamental and applied research. The complexity of cell cycle regulation and many examples of cellular differentiations in this bacterial group represent the main motives of this book. The book starts with discussing the regulation of cell cycle in alphaproteobacterial species from a system biology perspective. The following chapters specifically focus on the model species Caulobacter crescentus multiple layers of regulation, from transcriptional cascades to proteolysis and dynamic subcellular regulation of cell cycle regulators. In addition, the cell division process, chromosome segregation and growth of the cell envelope is described in detail. The last part of the book covers examples of non-Caulobacter alphaproteobacterial models, such as Agrobacterium tumefaciens, Brucella species and Sinorhizobium meliloti and also discusses possible applications. This book will be of interest to researchers in microbiology and cell biology labs working on cell cycle regulation and development.
Author: Morigen Morigen
Publisher: Frontiers Media SA
ISBN: 2889767671
Category : Science
Languages : en
Pages : 181
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Book Description
Analogous to the eukaryotic G1, S and M phase of the cell cycle, the bacterial cell cycle can be classified into independent stages. Slowly growing bacterial cells undergo three different stages, B-, C- and D-phase, respectively, while the cell cycle of fast-growing bacteria involves at least two independent cycles: the chromosome replication and the cell division. The oscillation in gene expression regulated by transcription factors, and proteolysis mediated by ClpXP, are closely correlated with progression of the cell cycle. Indeed, it has been shown that DnaA couples DNA replication initiation with the expression of the two oscillating regulators GcrA and CtrA, and the DnaA/GcrA/CtrA regulatory cascade drives the forward progression of the Caulobacter cell cycle. Furthermore, it has been found that: the DnaA oscillation in Eschericha coli and Caulobacter crescentus plays an important role in the cell cycle coordination; RpoS in Coxiella regulates the gene expression involved in the developmental cycle; the SigB and SinR transcription factors control whether cells remain in or leave a biofilm responding to metabolic conditions in Bacillus subtilis; similarly, BolA in most Gram-negative bacteria turns off motility and turns on biofilm development as a transcription factor; CtrA regulates cell division and outer membrane composition of the pathogen Brucella abortus; an essential transcription factor SciP enhances robustness of Caulobacter cell cycle regulation. Interestingly, transcription factors mediated metabolism fluctuations are also related to progression of the cell cycle. It has been shown that: CggR and Cra factors are involved in the flux-signaling metabolite fructose-1,6-bisphosphate; IclR mediates para-hydroxybenzoate catabolism in Streptomyces coelicolor; CceR and AkgR regulate central carbon and energy metabolism in alphaproteobacteria; and these metabolism changes affect cell growth. In line with the argument, AspC-mediated aspartate metabolism coordinates the E. coli cell cycle. However, the molecular mechanisms of maintaining the proper cell cycle progression through coordination of transcription factors mediated gene transcription oscillation, cellular metabolism with the cell cycle are not yet well-established. This Research Topic is intended to cover the spectrum of cell cycle regulatory mechanisms, in particular the coordination of transcription factor mediated gene transcription oscillations, and the cellular metabolisms associated with the cell cycle. We welcome all types of articles including Original Research, Review, and Mini Review. The subject areas of interest include but are not limited to: 1. Cell cycle coordination through gene expression and expression oscillation mediated by transcription factors. 2. Regulation of the cell cycle by proteolysis oscillation. 3. Coordination of the cell cycle with metabolism fluctuation. 4. DNA methylation fluctuation and the cell cycle. 5. Novel transcription factors and gene expression patterns associated with the cell cycle.
Author: Morigen Morigen
Publisher: Frontiers Media SA
ISBN: 2889743241
Category : Science
Languages : en
Pages : 193
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Book Description
Author: Jean-Pierre Tassan
Publisher: Springer
ISBN: 3319531506
Category : Science
Languages : en
Pages : 421
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Book Description
This book provides readers with an overview of the frequent occurrence of asymmetric cell division. Employing a broad range of examples, it highlights how this mode of cell division constitutes the basis of multicellular organism development and how its misregulation can lead to cancer. To underline such developmental correlations, readers will for example gain insights into stem cell fate and tumor growth. In turn, subsequent chapters include descriptions of asymmetric cell division from unicellular organisms to humans in both physiological and pathological conditions. The book also illustrates the importance of this process for evolution and our need to understand the background mechanisms, offering a valuable guide not only for students in the field of developmental biology but also for experienced researchers from neighboring fields.
